Notch signaling enhances nestin expression in gliomas

Abstract

Recent findings suggest that Notch signaling is active in brain tumors and stem cells, and that stem cells or cells with progenitor characteristics contribute to brain tumor formation. These stem cells are marked by expression of several markers, including nestin, an intermediate filament protein. We have studied how the Notch signaling pathway affects nestin expression in brain tumors. We find that Notch receptors and ligands are expressed in vitro and in human samples of glioblastomas, the highest grade of malignant gliomas. In culture, Notch activity activates the nestin promoter. Activation of the Notch pathway also occurs in a glioblastoma multiforme mouse model induced by Kras, with translational regulation playing a role in Notch expression. Combined activation of Notch and Kras in wild-type nestin-expressing cells leads to their expansion within the subventricular zone and retention of proliferation and nestin expression. However, activation of Notch alone is unable to induce this cellular expansion. These data suggest that Notch may have a contributing role in the stem-like character of glioma cells.

Figure 1

Notch receptor and ligand expression in human gliomas. (A) Analysis of Jagged1 mRNA expression in glioblastomas (GBMs) and non-GBM gliomas (NGGs) compared to normal brain. Data were derived from the microarray of Tanwar et al. [32]. (B) Western blot analysis of Notch receptors 1 and 2, ligands Delta-like1 and Jagged1, and corresponding actin bands in glial tumors.

Figure 2

Notch receptor and ligand expression in mouse glioma models. (A) Dark-field image of antisense Notch1 in situ hybridization, and hematoxylin and eosin (H&E) image of the same section in the cerebellum. Positive signal corresponds to a previously described pattern for Notch1 [34]. (B) Notch1 sense strand in situ hybridization control in tumor tissues. (C) Antisense in situ expression of Notch1, Delta-like1, Jagged1, and Hes1 mRNA in normal brain; PDGFB-induced oligodendroglioma; and Kras-induced spindle GBMs.

Figure 3

Semiquantitative analysis of Notch1 mRNA and protein in PDGFB and Kras tumors. (A) Sample dark-field images of Notch1 in situ in normal and tumor regions of PDGFB and Kras tumors. (B) Image analysis of positive signal regions in tumors. Positive pixel area (white grains in A) normalized to positive pixel areas in control normal brain images (*P < .01). (C) Western blot analysis of Notch1 and ligand expressions in PDGFB-induced oligodendrogliomas and Kras-induced spindle GBMs.

Figure 4

Notch1 activation of the nestin second intron element. (A) Alignment of human and mouse Nes second intron segments. Boxed regions indicate potential CBF-1-binding site. Numbering based on GenBank sequences: human AF004335 and mouse AY438043. (B and C) Assay for β-galactosidase activity. U251 cells were transfected with a nestin-β-galactosidase reporter, along with either (B) empty vector or (C) NICD-expressing vector. (D) Luciferase assay of U251 cells transfected with nestin reporter along with either empty vector or NICD-expressing vector (*P < .01). (E) Nestin IHC in Kras-induced spindle tumor in Ntv-a arf^-/- mouse. (F) Nestin IHC in PDGFB-induced oligodendroglioma.

Figure 5

Activated Notch and Kras can induce lesions in Ntv-a-targeted mice. (A) HES1 promoter luciferase assay with empty plasmid control or RCAS NICD transfection. (B) Table of lesion incidence in NICD/NICD + control virus, NICD + Akt, and NICD + Kras infections of Ntv-a mice (*P < .05). NICD + Kras compared to NICD/NICD + control virus. (C) H&E of NICD + Kras lesion located in the SVZ. (D) Kras + NICD lesion H&E, myc tag IHC of myc-tagged NICD, nestin IHC, and PCNA IHC. (E) HA Western blot analysis of cell lysates from rDelta1-HA-transfected and control-transfected cells. (F) IHC of HA (rDelta1) and nestin expression from a Kras + rDelta1-HA-generated tumor in Gtv-a arf^-/- mice.